Spectrophotometric and kinetic studies on the interaction of antibiotic X5108, the N-methylated derivative of kirromycin, with elongation factor Tu from Escherichia coli.
The absorption spectrum of antibiotic X5108, the N-methylated derivative of kirromycin, has been found to be decreased in intensity on binding to elongation factor (EF)-Tu . GDP, EF-Tu . GTP, and nucleotide-free EF-Tu. This has allowed the binding of X5108 to be studied directly. In agreement with previous studies, a 1:1 stoichiometry is observed, with a dissociation constant of less than 1 microM. Identical results were obtained with all three EF-Tu species. The absorption spectrum of X5108 in increasing concentrations of isopropyl alcohol first intensifies and then decreases, 80% isopropyl alcohol giving the same spectrum as that of X5108 bound to EF-Tu. This result is interpreted as showing that the chromophoric moiety of X5108 is bound in a highly hydrophobic environment on EF-Tu. The rate of binding of X5108 to EF-Tu . GDP was measured using a stopped flow spectrophotometer. This rate was proportional to the concentration of X5108, giving a second order binding rate constant of 4.8 X 10(3) M-1 s-1. Since this is several orders of magnitude too slow for a diffusion-controlled reaction, the results are interpreted based on a two-step binding process. A half-time of about 10 min is calculated for the dissociation of X5108 from EF-Tu . GDP. The fact that X5108 bound to EF-Tu is not in rapid equilibrium with X5108 free in solution needs to be considered in studies on the effect of X5108 and kirromycin on partial reactions of protein biosynthesis.
D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D000899:Aurodox / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D010444:Peptide Elongation Factor Tu; D010445:Peptide Elongation Factors / Q000378:metabolism*; D011485:Protein Binding; D013056:Spectrophotometry, Ultraviolet
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D010445:Peptide Elongation Factors; D000899:Aurodox; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D010444:Peptide Elongation Factor Tu